Chromogenic substrates of sialidase and methods of making and using the same

ABSTRACT

The subject invention discloses materials and methods for the design, synthesis, and biochemical evaluation of chromogenic substrate compounds for sialidases of bacterial, viral, protozoa, and vertebrate (including humans) origin. These compounds are based upon N-acetylneuraminic acid glycosides. In particular, this invention provides a novel class of these compounds as chromogenic substrates of these sialidases which yield chromogenic products after reactions catalyzed by sialidase take place. Also provided are methods of making these substrate compounds, methods of diagnosis and prognosis of sialidase related diseases using these substrate compounds.

CROSS-REFERENCE TO A RELATED APPLICATION

The present application is a divisional of the U.S. patent applicationSer. No. 09/651,622, filed Aug. 30, 2000, now U.S. Pat. No. 6,512,100,which is a continuation of U.S. patent application Ser. No. 08/958,356,filed Oct. 27, 1997, abandoned, which are hereby incorporated byreference herein in their entireties, including any figures, tables, ordrawings.

The research related to this invention is in part supported by acontract from the University of Alabama at Birmingham as a grant fromthe US Defense Advanced Research Projects Agency, grant number MDA972-97-K-0002.

FIELD OF THE INVENTION

The current invention relates to the design, synthesis, and biochemicalevaluation of chromogenic substrate compounds for sialidases ofbacterial, viral, protozoa, and vertebrate (including humans) origin. Inparticular, this invention provides a novel class of effective compoundsas chromogenic substrates of these sialidases which yield chromogenicproducts after reactions catalyzed by sialidase take place. Alsoprovided are methods of making these substrate compounds, methods ofdiagnosis and prognosis of sialidase related diseases using thesesubstrate compounds.

BACKGROUND OF THE INVENTION

Sialidase (EC, 3.2.1.18, also known as neuraminidase, acyineuraminylhydrolase) is a protein enzyme produced by many organisms such asbacteria, viruses, protozoa, and vertebrates including humans (Hirst, G.K. [1941] Science 94:22-23). This class of enzymes catalyzes thehydrolysis of a terminal sialic acid which is linked to oligosaccharidesthrough an O-glycosidic bond. Crystal structure of sialidases showedthat the enzyme has a highly conserved active site centered in apropeller like β-sheet twirl (Crennell, S. J. et al. [1993] Proc. Natl.Acad. Sci. USA 90:9852-9856).

Sialidases perform many critical biological functions. In bacteria,sialidase helps bacterial adhesion to tissues, and provides additionalnutritional sources (Crennell, S. et al. [1994] Structure 2(6):535-544).In viruses, it helps the release of progeny viruses (Liu, C. et al.[1995] J. Virol. 69:1099-1106). In a parasite, Trypanosoma cruzi, asialidase (also known as trans-sialidase) removes sialic acids frominfected cells and decorates its own surface with these sialic acids. Inhumans, sialidases are involved in protein digestion, immune responses,and cell proliferation. Abnormal production of sialidases may lead toserious human diseases such as sialidosis or increased Pseudomonasaeruginosa infection in cystic fibrosis patients.

Since sialidases are associated with many diseases, a color-producingsubstrate of sialidase would be an excellent diagnostic or prognosticreagent for sialidase-related diseases. For instance, sialidase level iselevated in bacterial vaginosis (Briselden, A. M. et al. [1992] J. Clin.Microbiol. 30:663-666). Measurement of sialidase level in the vaginalsamples could be used to diagnose bacterial vaginosis. In periodontaldisease caused by bacterial infection, it has been shown that presenceof sialidase increases the colonization of harmful bacteria (Liljemark,W. F. et al. [1989] Caries Res. 23:141-145). The cell invasion form ofT. cruzi, Trypomastigote, expresses high levels of trans-sialidaseactivity; therefore, measurement of trans-sialidase level could be usedfor diagnosis of T. cruzi infection and for monitoring disease progress(Cross, G. A., G. B. Takle [1993] Annu. Rev. Microbiol. 47:385-411). Incystic fibrosis patients, Pseudomonas aeruginosa infection is one of theleading causes of death. Sialidase was shown to be involved in thedisease progress (Cacalano, G. et al. [1992] J. Clin. Invest.89:1866-1874). Sialidase is also related to the regulation of cellproliferation (Bratosin, D. et al. [1995] Glycoconj. J. 12:258-267), theclearance of plasma proteins (Bonten, E. et al. [1996] Genes & Devel.10:3156-3169), and the catabolism of gangliosides and glycoproteins(Gornati, R. et al. [1997] Mol. Cell Biochem. 166:117-124).

Currently, there is available a synthetic substrate of sialidase,4-methylumbelliferyl-B-acetyl-neuraminic acid (4-MUN) (Lentz, M. R., R.G. Webster, G. M. Air [1987] Biochemistry 26:5351-5358), which producesa product with characteristic fluorescence spectrum upon hydrolysis.This change of fluorescence spectrum can only be measured with aspecialized instrument (fluorospectrometer). The substrate compounds ofthe current invention produce a visible color change upon hydrolysis,which is highly advantageous in medical diagnostic applications.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the current invention relates to the design andsynthesis of novel chromogenic substrate compounds for sialidases. Inanother embodiment, the subject invention pertains to the use of thenovel chromogenic substrates in assays for the detection of sialidases.The sialidases which are detected using the procedures and compounds ofthe subject invention are of bacterial, viral, protozoa, and vertebrate(including human) origin. In a specific embodiment, the subjectinvention provides a novel class of compounds which are useful aschromogenic substrates of sialidases.

In one embodiment, the present invention provides chromogenic sialidasesubstrate compounds having the following formula:

wherein, R₁═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, NHC(O)R₆,NHC(O)OR₆,Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₂═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein R₄═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆,Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₅═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein R₃═NO₂, CHO, (CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂, where k is aninteger from 1 to 3, or

wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3.

Also provided are chromogenic sialidase substrate compounds having theformula of General Structure I, wherein, R₁═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₂ or R₄═H, R₆, OR₆, OC(O)R₇,NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂,OPO₃R, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆,or CN, where j is an integer from 0 to 3; wherein, R₃═H, R₆, OR₆,OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆) ₂,C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₂ or R₄═NO₂, CHO, (CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂, where kis an integer from 1 to 3, or

wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3.

Also provided are chromogenic sialidase substrate compounds having theformula of General Structure I, wherein, R₁ or R₅═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₂═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₃═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₄═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₁ or R₅═NO₂, CHO,(CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂, where k is an integer from 1 to 3;wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(Ch₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j), CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₅═H or (CH₂)_(k)CH₃, where k is an integer from 0 to 4;wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₃, OC(O)R₄, NO₂, NH₂, N(R₃)₂, Cl, Br, I, F, CHO, CO₂R₃,C(O)N(R₃)₂, C(N—OH)NH₂, OPO₃R₃, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₃, OSO₃R₃,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₃, or CN, where j is an integer from 0 to 3;wherein, R₂═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein R₃═H, C(CH₃)₃,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or (CH₂)_(m)CH₃, where m isan integer from 0 to 3; wherein, R₄═R₃, OR₃, or N(R₃)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₃, OC(O)R₄, NO₂, NH₂, N(R₃)₂, Cl, Br, I, F, CHO, CO₂R₃,C(O)N(R₃)₂, C(N—OH)NH₂, OPO₃R₃, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₃, OSO₃R₃,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₃, or CN, where j is an integer from 0 to 3;wherein, R₂═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂) _(m)CH₃, where m is an integer from 0 to 3; wherein R₃═H, C(CH₃)₃,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or (CH₂)_(m)CH₃, where m isan integer from 0 to 3; wherein, R₄═R₃, OR₃, or N(R₃)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₆═H, OR₈, OC(O))R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO,CO₂R₈, C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈,OSO₃R₈, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0to 3; wherein, R₇═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO,CO₂R₈, C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈,OSO₃R₈, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0to 3; wherein R₈═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃,or (CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₉═R₈, OR₈,or N(R₈)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₆═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, BR, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₇═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₈═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₉═R₈, OR₈, orN(R₈)₂.

The subject invention further pertains to analogs, salts, derivatives,and mixtures of the subject compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a. A red color change produced by the substrate compound 14 (a)no sialidase added (left), (b) with sialidase added (right).

FIG. 1 b. An orange color change produced by the substrate compound 11(a) no sialidase added (left), (b) with sialidase added (right).

FIG. 2—synthetic approaches for selected examples from General StructureI are summarized in this reaction scheme.

FIG. 3—synthetic approaches for selected examples from General StructureII are summarized in this reaction scheme.

FIG. 4—synthetic approaches for selected examples from GeneralStructures IIIa and IIIb are summarized in this reaction scheme.

FIG. 5—synthetic approaches for selected examples from GeneralStructures IVa and IVb are summarized in this reaction scheme.

FIG. 6—shows an overall scheme for the preparation of methylN-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3),methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4), and N-acetyl-2-O -(4-formylphenyl)-β-D-neuraminic acid (5).

FIG. 7—shows an overall scheme for the preparation ofN-acetyl-2-O-[4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid (6).

FIG. 8—shows an overall scheme for the preparation of4-hydroxy-2-methoxybenzaldehyde (8), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate (9), andN-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminic acid (10).

FIG. 9—shows an overall scheme for the preparation ofN-acetyl-2-O-[3-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid(11).

FIG. 10—shows an overall scheme for the preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (12) and N-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13).

FIG. 11—shows an overall scheme for the preparation ofN-acetyl-2-O-[2-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid(14).

FIG. 12—shows the overall scheme for preparation ofN-acetyl-2-O-(5-bromo-4-chloroindol-3-yl)-α-D-neuraminic acid (28).

DETAILED DISCLOSURE OF THE INVENTION

The subject invention pertains to materials and methods useful fordetecting sialidase. Sialidase is an enzyme known to be associated witha variety of pathological conditions. Sialidases are produced bybacteria, viruses, and protozoa; therefore, detecting the presence ofsialidase in a biological sample can be indicative of the presence ofthese microbes. In specific embodiments, the detection of sialidases canbe performed according to the subject invention in order to identifyvaginal and periodontal infections, as well as to detect Pseudomonasaeruginosa in cystic fibrosis patients.

The presence of sialidase is detected according to the subject inventionthrough the use of novel chromogenic substrate compounds. Thesecompounds advantageously provide a visible color change when acted uponby sialidase. Thus, these substrates, when utilized according to theteachings of the subject invention, can be used to easily and accuratelydetect the presence of sialidase in a sample. In a preferred embodiment,the sample which is tested is a biological sample such as blood, mucous,saliva, and the like.

The subject invention provides compounds having structures as shown inGeneral Structures I, II, IIIa, IIIb, IVa, and IVb. The inventionfurther includes derivatives, analogs, and salts of the exemplifiedcompounds. These derivatives, analogs, and salts, which can readily beprepared by one skilled in the art and having the benefit of the instantdisclosure, fall within the scope of the present invention so long assuch compounds have the characteristic of producing a color change whenacted upon by a sialidase enzyme.

The compounds of the subject invention can be employed in a wide varietyof assay formats. Typically, the assay will involve contacting a sampleto be tested for the presence of sialidase with a chromogenic enzymesubstrate of the subject invention. A color change occurring after thesample is contacted with the substrate is indicative of the presence ofsialidase. The assay may optionally utilize positive and/or negativecontrols to aid in the interpretation and verification of the results.The results may also be quantified using standard optical measuringinstrumentation.

Materials and Methods

Biochemical Evaluation for the Chromogenic Product of SialidaseSubstrate Compounds

Sialidase can be obtained from, for example, purified recombinantbacterial sialidase from Salmonella T., whole influenza virus, orculture medium containing secreted human sialidase from 2CFSME cellline. The sialidase preparation is added to a buffer of 0.1 M sodiumacetate at pH6.0, and the substrate compound is provided at about 0.5 mMconcentration. The reaction takes place in room temperature for 20 minsin a volume of 100 μl. At the end of the reaction, the pH is adjusted byadding a solution (0.2 M glycine, and sodium hydroxide with a pH valueof 11.0). A color change is readily visible as exemplified by FIGS. 1 aand 1 b. The color change can be quantitated by measuring the lightabsorption of the reaction mixture.

FIG. 1 a shows a red color change produced by the substrate compound 14(a) no sialidase added (left), (b) with sialidase added (right). FIG. 1b shows an orange color change produced by the substrate compound 11 (a)no sialidase added (left), (b) with sialidase added (right).

General Methodologies

The following general methods are applicable to the synthesis ofcompounds of the invention. Modifications or variations of these methodscan readily be utilized by those skilled in the art having the benefitof the instant disclosure.

Esterification

N-Acetyl-D-neuraminic acid is treated with methanol-washed Dowex 50W-X4in methanol with stirring at room temperature for a period of time,generally 4 h. The mixture is filtered, and the filtrate is concentratedto give the desired esterified product after crystallization.

Those skilled in the art would recognize that other standard proceduresare available for esterification of the same material, such as the useof other cation exchange resins, e.g., Amberlyst 15 or Dowex 50W-X8,among others.

O-Acetylation and Glycosyl Chloride Preparation

Treatment of the esterified product with acetyl chloride with stirringat room temperature under anhydrous conditions for a period of time,generally 20-24 h, results in formation of the per-O-acetylated glycosylchloride. Note that in some instances the bubbling of dry hydrogenchloride (gas) into the reaction vessel is necessary to effect glycosylchloride formation. Concentration of the reaction mixture with the waterbath temperature not exceeding 35° C., and drying the residue in vacuoprovides the product as a foam sufficiently pure for subsequentreactions.

Those skilled in the art would recognize that other standard proceduresare available for O-acetylation and glycosyl chloride preparation of thesame material, including a previously reported two-step procedure (Kuhn.et al., 1966) which involves per-O-acetylation of the same material withacetic anhydride in perchloric acid, followed by formation of theglycosyl chloride by treatment with acetyl chloride.

O-Glycosylation

Treatment of the substituted hydroxybenzaldehyde derivative with sodiumhydride in tetrahydrofuran with stirring at room temperature for aperiod of time, generally 1-3 h, results in formation of the sodiumsalt. Subsequent treatment of the sodium salt with the glycosyl chloride(compound 3) with stirring, for a period of time, generally 12-60 h, atroom temperature results in O-glycosylation. Concentration of thereaction mixture, treatment of the residue with ethyl acetate and water,separation and drying of the organic phase, concentration of the organicphase, and column chromatography of the crude material affords thedesired O-glycoside.

Those skilled in the art would recognize that other standard proceduresare available for O-glycosylation of the same materials, such astraditional Lewis Acid-mediated O-glycosylation methodologies (Okamotoand Goto, 1990), as well as the use of alternate salts of thesubstituted aromatic hydroxyl derivative, including tetrabutylammonium(Baggett and Marsden, 1982) or silver (Holmquist and Brossmer, 1972)salts, among others.

De-O-acetylation and De-esterification

The protected O-glycoside is taken up in aqueous sodium hydroxide andstirred at room temperature for a period of time, generally 1-4 h. Themixture is then adjusted to pH 3-5 with Dowex 50W-X4 (H+) resin.Filtration, followed by lyophilization of the filtrate affords thedesired de-O-acetylated and de-esterified material.

Those skilled in the art would recognize that other standard proceduresare available for the complete de-O-acetylation and de-esterification ofthe same material, including a two-step procedure which involvescomplete de-O-acetylation of the same material with sodium methoxide inmethanol or with an appropriate ion exchange resin, e.g. AmberliteIRA-400 (OH−), followed by de-esterification using conditions of acidhydrolysis or base hydrolysis.

Synthesis of Chromogenic Substrates of Sialidases

A. Compounds with General Structure I and their salts and derivatives,may be prepared using any of several methods known in the art for thesynthesis of substituted sialic acid analogs containing analogousstructures.

To illustrate, synthetic approaches for selected examples (FIG. 2) fromGeneral Structure I are summarized in the following reaction scheme andare representative of the types of procedures which can be employed.Table 1 lists specific compounds, the synthesis of which is exemplifiedherein.

TABLE 1

Compound R₁ R₂ R₃ 5 H H CHO 6 H H CH═CHNO₂ 10 H OCH₃ CHO 11 H OCH₃CH═CHNO₂ 13 OCH₃ H CHO 14 OCH₃ H CH═CHNO₂

In another specific embodiment, the subject invention includes compoundshaving the following structures:

Advantageously, these compounds produce a blue color change when actedupon by a sialidase.

FIG. 2 illustrates constructing a basic skeleton of General Structure Ivia acid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofcompound 3 with the sodium salt of numerous substitutedhydroxybenzaldehyde derivatives would provide the key intermediates tothe desired targets (compounds 4, 9, and 12). Generation of the sodiumsalt would be accomplished with sodium hydride in tetrahydrofuran. Thismethod of O-glycosylation has already been applied in thestereoselective preparation of numerous -O-glycosides ofN-acetyl-D-neuraminic acid (Myers, et al. , 1980; Eschenfelder andBrossmer Carbohydr. Res., 1987; Eschenfelder and Brossmer,Glycoconjitgate J., 1987; Okamoto and Goto, 1990; Warner and O'Brien,1979) derived from aromatic hydroxyls. However, none of the productsdescribed herein are contained in the aforementioned references.Synthetic approaches to or references to synthetic approaches tointermediates (1) and (2) are contained in the aforementionedreferences. Subsequent de-O-acetylation and de-esterification of theresulting intermediates can be accomplished with an aqueous sodiumhydroxide solution and workup involving acidification of the reactionmedium. This provides access to the formyl substitutedphenolic-O-glycosides (compounds 5, 10, and 13).

Treatment of the derived targets (compounds 5, 10, and 13) withnitromethane, ammonium acetate, and acetic acid in ethanol under refluxprovides access to the desired nitrovinyl targets (compounds 6, 11, and14). This procedure has been utilized in the preparation of nitrovinylanalogs of other monosaccharides (Patel and Richardson, 1986; Aamlid, etal., 1990) as chromogenic substrates for the assay of glycosidases;however, none of the products or intermediates described herein arecontained in the aforementioned references.

It should also be noted that the p-nitrophenyl O-glycoside ofN-acetylneuraminic acid (General Structure I, wherein, R₁═R₂═R₄═R₅═H andR₃═NO₂ has been reported as a chromogenic substrate of sialidases(Eschenfelder and Brossmer, Carbohydr. Res., 1987). Condensation ofcompounds 5, 10, or 13 with any of numerous aromatic keto compounds inthe presence of ammonia and ammonium chloride, provides ready access tonumerous chromogenic substrates of sialidases (for representativeexamples, see compounds 15 and 16 FIG. 2) of General Structure I.

B. Compounds with General Structure II and their salts and derivatives,may be prepared using any of several methods known in the art for thesynthesis of substituted sialic acid analogs containing analogousstructures.

To illustrate, synthetic approaches for selected examples from GeneralStructure II (FIG. 3) are summarized in the following reaction schemeand are representative of the types of procedures to be employed. FIG. 3illustrates constructing a basic skeleton of General Structure II viaacid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofany of numerous substituted indoxyl 1,3-diacetate compounds (compound17) with sodium methoxide in anhydrous N,N-dimethylformamide readilyprovides the modified 3-hydroxy indole (compound 18). This procedure hasbeen utilized in the preparation of 5-bromo-3-hydroxyindole (compound18, wherein, R₁═R₃═R₄═H and R₂═Br) (Eschenfelder and Brossmer,Glycoconjugate J., 1987). Subsequent treatment of the compound 18 withcompound 3 in anhydrous N,N-dimethylformamide provides the desiredmodified indole O-glycoside (compound 19) according to a known procedurefor the preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(5-bromoindol-3-yl)-α-D-neuraminate(compound 19, wherein, R₁═R₃═R₄═R₅═H and R₂═Br) (Eschenfelder andBrossmer, Glycoconjugate J., 1987). Analogously, 3-indolyl O-glycosidesof other monosaccharides have been prepared using these and alternateconditions (Robertson, 1927; Freudenberg, et al., 1952; Anderson andLeeback, 1961; Horwitz, et al., 1964; Ley, et al., 1987); however, noneof the products or intermediates described herein are contained in theaforementioned references. Treatment of compound 19 with sodium hydridein tetrahydrofuran, followed with an alkyl halide (R₅Br) would providethe N-alkylated product. Subsequent de-O-acetylation andde-esterification of the resulting intermediates can be accomplishedwith an aqueous sodium hydroxide solution and workup involvingacidification of the reaction medium. This provides access to thesubstituted indole -O-glycosides (compound 20). It should be noted thatN-acetyl-2-O-(5-bromoindol-3-yl)-α-D-neuraminic acid (compound 20,wherein, R₁═R₃═R₄═R₅═H and R₂═Br) has been utilized as a chromogenicsubstrate for sialidases of many different origins (Eschenfelder andBrossmer, Glycoconjugate J., 1987).

C. Compounds with General Structures IIIa and IIIb and their salts andderivatives, may be prepared using any of several methods known in theart for the synthesis of substituted sialic acid analogs containinganalogous structures.

To illustrate, synthetic approaches for selected examples from GeneralStructures IIIa and IIIb are summarized in FIG. 4 and are representativeof the types of procedures to be employed. FIG. 4 illustratesconstructing a basic skeleton of General Structures IIIa/IIIb viaacid-mediated esterification of commercially, availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofcompound (3) with the sodium salt of numerous substituted coumarinderivatives provides the key intermediates to the desired targets(compound 21). Generation of the sodium salt can be accomplished withsodium hydride in tetrahydrofuran. This method of O-glycosylation hasalready been applied in the stereoselective preparation of numerous-O-glycosides on N-acetyl-D-neuraminic acid (Myers, et al., 1980;Eschenfelder and Brossmer, Carbohydr. Res., 1987; Eschenfelder andBrossmer, Glycoconjugate J., 1987; Okamoto and Goto, 1990; Warner andO'Brien, 1979) derived from aromatic hydroxyls, including specificexamples for the preparation of a substituted coumarin O-glycoside(compound 21, wherein, R₁═H and R₂═CH₃) (Warner and O'Brien, 1979;Myers, et al., 1980). Subsequent de-O-acetylation and de-esterificationof the resulting intermediates can be accomplished with an aqueoussodium hydroxide solution and workup involving acidification of thereaction medium. This provides access to the modified coumarinO-glycosides (compound 22).

D. Compounds with General Structures IVa and IVb and their salts andderivatives, may be prepared using any of several methods known in theart for the synthesis of substituted sialic acid analogs containinganalogous structures.

To illustrate, potential synthetic approaches for selected examples fromGeneral Structures IVa and IVb are summarized in FIG. 5 and arerepresentative of the types of procedures which can be employed. FIG. 5illustrates constructing a basic skeleton of General Structure IVa/IVbvia acid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofcompound (3) with the sodium salt of numerous substituted naphtholderivatives would provide the key intermediates to the desired targets(compound 23). Generation of the sodium salt can be accomplished withsodium hydride in tetrahydrofuran. This method of O-glycosylation hasalready been applied in the stereoselective preparation of numerous-O-glycosides on N-acetyl-D-neuraminic acid (Myers, et al., 1980;Eschenfelder and Brossmer, Carbohydr. Res., 1987; Eschenfelder andBrossmer, Glycoconjugate J., 1987; Okamoto and Goto, 1990; Warner andO'Brien, 1979) derived from aromatic hydroxyls. However, none of theproducts described herein are contained in the aforementionedreferences. Synthetic approaches to, or references to syntheticapproaches to, intermediates (1) and (2) are contained in theaformentioned references. Subsequent de-O-acetylation andde-esterification of the resulting intermediates can be accomplishedwith an aqueous sodium hydroxide solution and workup involvingacidification of the reaction medium. This would provide access to themodified naphthyl O-glycosides (compounds 24).

F. Biochemical Evaluation for the Chromogenic Product of the SialidaseSubstrate Compound

The source of sialidase was from purified recombinant bacterialsialidase from Salmonella T., whole influenza virus, or culture mediumcontaining secreted human sialidase from 2CFSME0 cell line. Thesialidase preparation was added to a buffer of 0.1 M sodium acetate atpH6.0, and the substrate compound 14 was provided at about 0.5 mMconcentration. The reaction took place in room temperature for 20 minsin a volume of 100 μl. At the end of the reaction, the pH was adjustedby adding a solution (0.2 M glycine, and sodium hydroside with a pHvalue of 11.0). A color change to red was readily visible as exemplifiedby FIGS. 1 a and 1 b. The color change was quantitated by measuring thelight absorption of the reaction mixture. The light absorption wasscanned with a photospectrometer. The peak value for compound IBX4010 is495 nm. At a substrate concentration of 0.2 mM, the light absorption at495 nm with a 1 cm path is 1.203. The control in which the reactionmixture was kept under the same condition for 10 minutes withoutaddition of any enzyme had an absorption of 0.282 at 495 nm with a 1 cmpath.

Compound 11 was tested by the same method. At the end of the reactionwith pH adjusment, a color change to orange was readily visible asexemplified by FIGS. 1 a and 1 b. The color change was quantitated bymeasuring the light absorption of the reaction mixture. Ten minutesafter the reaction, the mixture of the reaction product was adjusted tobasic pH and the light absorption was scanned with a photospectrometer.The peak value for compound IBX4023 is 480 nm. At a substrateconcentration of 0.2 mM, the light absorption at 480 nm with a 1 cm pathis 4.065. The control in which the reaction mixture was kept under thesame condition for 10 minutes without addition of any enzyme had anabsorption of 1.452 at 480 nm with a 1 cm path.

G. Classes of Chromogenic Substrate Compounds of Sialidases

As used herein, the “effective amount” of a compound of the inventionrequired for the use in the method presented herein will differ not onlywith the particular compound to be selected but also with the mode ofapplication, and the nature of the sample specimen. The exact amountwill be evaluated by testing with a sufficient number of clinicalsamples in each application as conducted by persons skilled in the art.However, a generally suitable concentration will range from about 0.1 toabout 10 mM/ml of testing solutions. Furthermore, the compounds may beused as pure chemical applied to a test solution, or as a purechemically acceptable salt or derivative. However, it is preferable toprovide the active chemical or its chemically acceptable salt orderivative, as a medicinal formulation, either as a dry material(reaction solution provided separately), or as a solution or suspension(an aqueous solution or other chemically acceptable solvent solutions),or as a dip stick. The subject specimen can be applied to the test formeasuring the activity levels of sialidases. Those skilled in the arthaving the benefit of the instant disclosure will appreciate thatamounts and modes of application are readily determinable without undueexperimentation.

The following detailed examples for methods of preparation are forillustration only, and are not intended to represent a limitation of theinvention. The structures of the compounds whose preparations aredescribed below are summarized in Table 1 for modified phenolderivatives and in FIG. 3 (for a single example where R₁═Cl; R₂═Br;R₃═R₄═R₅═H). In all cases synthetic intermediates and products werefound to be pure according to standards known to those skilled in theart (such as thin layer chromatography, melting or boiling points, gaschromatography, ion exchange chromatography, and/or high pressure liquidchromatography, elemental analysis, and spectroscopic methods).Furthermore, structures were characterized and assigned by spectroscopicmethods considered standard practices by those skilled in the art (suchas infrared, ultraviolet, and mass spectroscopies, ¹H and ¹³C nuclearmagnetic resonance spectroscopy, and/or x-ray crystallography). Selectedspectral data are described for intermediates and products.

EXAMPLE 1

Preparations of Methyl N-acetyl-β-D-neuraminate (2), MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3),methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4), and N-acetyl-2-O-(4-formylphenyl)-α-D-neuraminic acid (5)

The overall scheme is shown in FIG. 6.

Preparation of Methyl N-acetyl-β-D-neuraminate (2)

To a stirred suspension of N-acetylneurarninic acid (1) (10.0 g, 32.3mmol) in methanol (1.0 L) was added methanol-washed Dowex 50W-X4 (25.0g) under a nitrogen atmosphere at room temperature protected from light.The resulting mixture was allowed to stir at room temperature for 4 h.The mixture was filtered and the filtrate was concentrated to dryness.The residue was crystallized from methanol to afford pure compound (2)(10.1 g, 96%): mp 178-180° C. (d). A literature reference (Kuhn et al.,1966) reports mp 179-180° C. A second literature reference (Ogura etal., 1986) reports mp 180-182° C.

¹H NMR (D₂O): (1.73 (dd, 1 H, J_(3a,4) 12.0 Hz, J_(3a,3e) 13.3 Hz,H-3a), 1.87 (s, 3 H, NAc), 2.14 (dd, 1 H, J_(3e,4) 5.0 Hz, H-3e),3.33-3.48 (m, 2 H), 3.52-3.58 (m, 1 H), 3.62-3.68 (m, 1 H), 3.65 (s, 3H, CO₂CH₃), 3.69-3.76 (m, 1 H), 3.84-3.92 (m, 2 H).

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3)

A suspension of compound (2) (3.67 g, 11.3 mmol) in acetyl chloride (225mL) was stirred under anhydrous conditions at room temperature protectedfrom light for 24 h. The resulting solution was concentrated to dryness,the residue was coevaporated with anhydrous ether (2×50 mL), followed bycoevaporations with anhydrous benzene (2×50 mL). Note that in allevaporations, the water bath temperature was maintained at or below 35°C. The residue was dried in vacuo to afford pure compound (3) (4.8 g,83%) as a syrup. A literature reference (Ogura et al., 1986) reports mp116-118° C.; whereas, a second literature reference (Kuhn et al., 1966)reports compound (3) as a syrup.

¹H NMR (CDCl₃): (1.92 (s, 3 H, NAc), 2.06, 2.07, 2.10, 2.14 (4 s, 12 H,4 X OAc), 2.28 (dd, 1 H, J_(3a,4) 11.3 Hz, J_(3a,3e) 13.5 Hz, H-3a),2.79 (dd, 1 H, J_(3e,4) 4.5 Hz, H-3e), 3.89 (s, 3 H, CO₂CH₃), 4.07 (dd,1 H, J_(8,9′) 5.6 Hz, J_(9′,9″) 11.6 Hz, H-9′), 4.13-4.28 (m, 1 H, H-5),4.36 (dd, 1 H, J_(6,7) 2.5 Hz, J_(5,6) 10.5 Hz, H-6), 4.43 (dd, 1 H,J_(8,9″) 2.9 Hz, H-9″), 5.18 (ddd, 1 H, J_(7,8) 6.7 Hz, H-8), 5.40 (ddd,1 H, J_(4,5) 10.4 Hz, H-4), 5.49-5.52 (m, 2 H, H-7, NH).

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate (4)

To a stirred solution of 4-hydroxybenzaldehyde (121 mg, 0.98 mmol) inanhydrous tetrahydrofuran (6.0 mL) was added portionwise sodium hydride(48 mg of a 60% dispersion in mineral oil, 1.2 mmol) under a nitrogenatmosphere at room temperature. The resulting mixture was allowed tostir at room temperature for 25 min. The mixture was treated withcompound (3) (500 mg, 0.98 mmol) and the resulting mixture was stirredunder a nitrogen atmosphere at room temperature for 52 h. The mixturewas concentrated to dryness, the residue was diluted with ethyl acetate(15 mL), and washed with water (15 mL). The aqueous phase was extractedwith ethyl acetate (3×15 mL), and combined organic phases were driedwith magnesium sulfate, filtered, and the filtrate was concentrated todryness. The residue was chromatographed (silica gel, 1:1acetone-hexanes as eluting solvent) to afford pure compound (4) (238 mg,41%): R_(ƒ)=0.26 (1:1 acetone-hexanes; UV, H₂SO₄).

¹H NMR (CDCl₃): (1.95 (s, 3 H, NAc), 2.07, 2.09, 2.13, 2.22 (4 s, 12 H,4 X OAc), 2.32 (ut, 1 H, J_(3a,3e)=J_(3a,4)=13.2 Hz, H-3a), 2.77 (dd, 1H, J_(3e,4) 5.0 Hz, H-3e), 3.67 (s, 3 H, CO₂CH₃), 4.10-4.22 (m, 2 H),4.24-4.32 (m, 1 H), 4.63 (dd, 1 H, J 2.0 Hz, J 11.7 Hz), 4.97-5.06 (m, 1H), 5.30 (d, 1 H, J 12.6 Hz), 5.41 (s, 2 H), 7.20 (d, 2 H, J 9.6 Hz, 2 XArH), 7.86 (d, 2 H, J 9.6 Hz, 2 X ArH), 9.95 (s, 1 H, CHO).

Preparation of N-Acetyl-2-O-(4-formylphenyl)-α-D-neuraminic acid (5)

A solution of compound (4) (171 mg, 0.29 mmol) in aqueous sodiumhydroxide (5.0 mL of a 1.0 M solution, 5.0 mmol) was stirred at roomtemperature for 2 h. The resulting mixture was cooled to 0° C. andtreated with methanol-washed Dowex 50W-X4 til pH 3. The mixture wasfiltered, the filtered resin was rinsed with water, and the filtrate waslyophilized to afford compound (5) (118 mg, 99%): R_(ƒ)=0.31 (5:2:1ethyl acetate-methanol-0.02% aqueous calcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.08 (s, 3 H, NAc), 2.05-2.12 (m, 1 H, H-3a), 2.84 (dd, 1H, J_(3e,4) 5.6 Hz, J_(3a,3e) 13.1 Hz, H-3e), 3.58-3.69 (m, 2 H),3.82-3.90 (m, 3 H), 3.92-4.10 (m, 1 H), 4.21 (dd, 1 H, J 1.9 Hz, J 11.3Hz), 7.32 (d, 2 H, J 9.6 Hz, 2 X ArH), 7.92 (d, 2 H, J 9.6 Hz, 2 X ArH),9.84 (s, 1 H, CHO).

EXAMPLE 2

Preparation of N-acetyl-2-O-[4-(2-nitroyinyl)phenyl]-(α-D-neuraminicacid (6)

The overall reaction scheme is shown in FIG. 7. For the preparations ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3),methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4), and N-acetyl-2-O-(4-formylphenyl)-α-D-neuraminic acid (5), see theexperimental details for Example 1.

To a stirred solution of compound (5) (50 mg, 0.10 mmol) in a mixture ofethanol (2.0 mL) and acetic acid (0.05 mL) was added ammonium acetate(50 mg, 0.65 mmol) and nitromethane (0.20 mL, 3.70 mmol) at roomtemperature. The reaction mixture was heated under reflux for 30 min,cooled to room temperature, and evaporated to dryness. The residue waschromatographed (silica gel, 5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride as eluting solvent) to afford pure compound (6) (32 mg,68%): R_(ƒ)=0.50 (5:2:1 ethyl acetate-methanol-0.02% aqueous calciumchloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.05 (s, 3 H, NAc), 1.95-2.02 (m, 1 H, H-3a), 2.89 (dd, 1H, J_(3e,4) 5.2 Hz, J_(3a,3e) 12.9 Hz, H-3e), 3.57-3.69 (m, 2 H),3.85-4.00 (m, 4 H), 4.06 (dd, 1 H, J 1.5 Hz, J 10.5 Hz), 7.22 (d, 2 H, J9.0 Hz, 2 X ArH), 7.64 (d, 2 H, J 9.0 Hz, 2 X ArH), 7.83 (d, 1 H, J 13.5Hz, H-vinylic), 8.13 (d, 1 H, J 13.5 Hz, H-vinylic).

EXAMPLE 3

Preparation of 4-hydroxy-2-methoxybenzaldehyde (8), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9), and N-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminic acid(10)

The overall reaction scheme is shown in FIG. 8.

For the preparation of methyl N-acetyl-α-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

Preparation of 4-Hydroxy-2-methoxybenzaldehyde (8)

To a stirred solution of 3-methoxyphenol (14.9 g, 120 mmol) in 15%aqueous potassium hydroxide (500 mL) was added chloroform (100 mL). Theresulting solution was heated under reflux for 4 h, cooled to roomtemperature, and treated with 10% aqueous hydrochloric acid til pH 4.The suspension was filtered, and the filter cake was rinsed withchloroform (150 mL). The chloroform phase was separated, the aqueousphase was extracted with additional portions of chloroform (3×50 mL),and the combined organic phases were dried with magnesium sulfate. Thesolution was then filtered through a short column (silica gel,chloroform as eluting solvent) to afford compound (8). Crystallizationfrom ethyl acetate gave pure compound (8) (1.8 g, 10%): mp 150-152° C. Aliterature reference (Patel and Richardson, 1986) reports mp 154-156° C.Additional references (Tiemann and Koppe, 1881; de Kiewiet and Stephen,1931) report mp 153° C.

¹H NMR (CDCl₃): (3.87 (s, 3 H, OCH₃), 6.32-6.48 (m, 2 H, 2 X ArH), 7.62(d, 1 H, J 9.6 Hz, ArH), 10.1 (s, 1 H, CHO).

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9)

To a stirred solution of compound (8) (900 mg, 5.92 mmol) in anhydroustetrahydrofuran (35 mL) was added portionwise sodium hydride (288 mg ofa 60% dispersion in mineral oil, 7.2 mmol) under a nitrogen atmosphereat room temperature. The resulting mixture was allowed to stir at roomtemperature for 2.5 h. The mixture was treated with compound (3) (2.33g, 4.58 mmol) and the resulting mixture was stirred under a nitrogenatmosphere at room temperature for 115 h. The mixture was concentratedto dryness, the residue was diluted with ethyl acetate (40 mL), andwashed with water (40 mL). The aqueous phase was extracted with ethylacetate (3×40 mL), and combined organic phases were dried with magnesiumsulfate, filtered, and the filtrate was concentrated to dryness. Theresidue was chromatographed (silica gel, 1:1 acetone-hexanes as elutingsolvent) to afford pure compound (9) (1.23 g, 43%): R_(ƒ)=0.31 (1:1acetone-hexanes; UV, H₂SO₄).

¹H NMR (CDCl₃): (1.96 (s, 3 H, NAc), 2.07, 2.09, 2.14, 2.18 (4 s, 12 H,4 X OAc), 2.23-2.45 (m, 1 H, H-3a), 2.74 (dd, 1 H, J_(3e,4) 5.9 Hz,J_(3a,3e) 13.7 Hz, H-3e), 3.73 (s, 3 H, CO₂CH₃), 3.93 (s, 3 H, OCH₃),4.12-4.22 (m, 2 H), 4.25-4.30 (m, 1 H), 4.60 (dd, 1 H, J 1.8 Hz, J 12.6Hz), 4.96-5.08 (m, 1 H), 5.28-5.47 (m, 3 H), 6.65 (d, 1 H, J 3.0 Hz,ArH), 6.74 (dd, 1 H, J 3.0 Hz, J 9.6 Hz, ArH), 7.82 (d, 1 H, J 9.6 Hz,ArH), 10.33 (s, 1 H, CHO).

Preparation of N-Acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminicacid (10)

A solution of compound (9) (751 mg, 1.20 mmol) in aqueous sodiumhydroxide (20.0 mL of a 1.0 M solution, 20.0 mmol) was stirred at roomtemperature for 2 h. The resulting mixture was cooled to 0° C. andtreated with methanol-washed Dowex 50W-X4 til pH 3. The mixture wasfiltered, the filtered resin was rinsed with water, and the filtrate waslyophilized to afford compound (10) (288 mg, 54%): R_(ƒ)=0.34 (5:2:1ethyl acetate-methanol-0.02% aqueous calcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.08 (s, 3 H, NAc), 2.04-2.12 (m, 1 H, H-3a), 2.87 (dd, 1H, J_(3,4) 5.6 Hz, J_(3a,3e) 15.0 Hz, H-3e), 3.60-3.68 (m, 2 H),3.81-3.95 (m, 4 H), 3.93 (s, 3 H, OCH₃), 4.23 (dd, 1 H, J 3.7 Hz, J 11.2Hz), 6.85 (dd, 1 H, J 5.7 Hz, J 11.4 Hz, ArH), 6.97 (d, 1 H, J 5.7 Hz,ArH), 7.75 (d, 1 H, J 11.4 Hz, ArH), 10.0 (s, 1 H, CHO).

EXAMPLE 4

Preparation ofN-acetyl-2-O-[3-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid (11)

The overall reaction scheme is shown in FIG. 9. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

For the preparations of 4-hydroxy-2-methoxybenzaldehyde (8), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9), and N-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminic acid(10), see the experimental details presented in Example 3.

To a stirred solution of compound (10) (120 mg, 0.27 mmol) in a mixtureof ethanol (4.8 mL) and acetic acid (0.12 mL) was added ammonium acetate(120 mg, 1.56 mmol) and nitromethane (0.48 mL, 8.86 mmol) at roomtemperature. The reaction mixture was heated under reflux for 30 min,cooled to room temperature, and evaporated to dryness. The residue waschromatographed (silica gel, 5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride as eluting solvent) to afford pure compound (11) (48mg, 36%): R_(ƒ)═0.64 (5:2:1 ethyl acetate-methanol-0.02% aqueous calciumchloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.06 (s, 3 H, NAc), 1.98-2.03 (m, 1 H, H-3a), 2.88 (dd, 1H, J_(3e,4)5.1 Hz, J_(3a,3e) 14.0 Hz, H-3e), 3.59-3.68 (m, 2 H),3.75-4.00 (m, 4 H), 3.95 (s, 3 H, OCH₃), 4.11 (dd, 1 H, J 2.5 Hz, J 11.4Hz), 6.82 (d, 1 H, J 10.8 Hz, ArH), 6.95 (br s, 1 H, ArH), 7.54 (d, 1 H,J 10.8 Hz, ArH), 8.00 (d, 1 H, J 13.5 Hz, H-vinylic), 8.22 (d, 1 H, J13.5 Hz, H-vinylic).

EXAMPLE 5

Preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (12) and N-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13)

The overall reaction scheme is shown in FIG. 10. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (12)

To a stirred solution of vanillin (4-hydroxy-3-methoxybenzaldehyde) (273mg, 1.8 mmol) in anhydrous tetrahydrofuran (12.0 mL) was addedportionwise sodium hydride (86 mg of a 60% dispersion in mineral oil,2.2 mmol) under a nitrogen atmosphere at room temperature. The resultingmixture was allowed to stir at room temperature for 2.5 h. The mixturewas treated with compound (3) (700 mg, 1.38 mmol) and the resultingmixture was stirred under a nitrogen atmosphere at room temperature for68 h. The mixture was concentrated to dryness, the residue was dilutedwith ethyl acetate (25 mL), and washed with water (25 mL). The aqueousphase was extracted with ethyl acetate (3×25 mL), and combined organicphases were dried with magnesium sulfate, filtered, and the filtrate wasconcentrated to dryness. The residue was chromatographed (silica gel,chloroform, followed by ethyl acetate as eluting solvent) to afford purecompound (12) (322 mg, 38%): R_(ƒ)=0.64 (1:8 acetone-ethyl acetate; UV,H₂SO₄).

¹H NMR(CDCl₃):(1.94 (s, 3 H, NAc), 2.08, 2.09, 2.14, 2.19 (4 s, 12 H, 4X OAc), 2.33 (ut, 1 H, J _(3a,3e) =J_(3a,4)=13.5 Hz, H-3a), 2.82 (dd, 1H, J_(3e,4) 5.4 Hz, H-3e), 3.70 (s, 3 H, CO₂CH₃), 3.92 (s, 3 H, OCH₃),4.10-4.18 (m, 2 H), 4.23-4.31 (m, 1 H), 4.52 (br d, 1 H, J 11.4 Hz),4.97-5.12 (m, 1 H), 5.20-5.28 (m, 1 H), 5.30-5.40 (m, 2 H), 7.32 (d, 1H, J 9.0 Hz, ArH), 7.41-7.48 (m, 2 H, 2 X ArH), 9.92 (s, 1 H, CHO).

Preparation of N-Acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13)

A solution of compound (12) (236 mg, 0.38 mmol) in aqueous sodiumhydroxide (6.0 mL of a 1.0 M solution, 6.0 mmol) was stirred at roomtemperature for 160 min. The resulting mixture was cooled to 0° C. andtreated with methanol-washed Dowex 50W-X4 til pH 3. The mixture wasfiltered, the filtered resin was rinsed with water, and the filtrate waslyophilized to afford compound (13) (152 mg, 91%): R_(ƒ)=0.46 (5:2:1ethyl acetate-methanol-0.02% aqueous calcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.08 (s, 3 H, NAc), 2.03-2.12 (m, 1 H, H-3a), 2.92 (dd, 1H, J_(3e,4) 5.2 Hz, J_(3a,3e) 13.9 Hz, H-3e), 3.54-3.70 (m, 2 H),3.80-4.20 (m, 5 H), 3.92 (s, 3 H, OCH₃), 7.48 (d, 1 H, J 9.6 Hz, ArH),7.52-7.60 (m, 2 H, 2 X ArH), 9.82 (s, 1 H, CHO).

EXAMPLE 6

Preparation ofN-acetyl-2-O-[2-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid (14)

The overall reaction scheme is shown in FIG. 11. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

For the preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (12) and N-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13), see the experimental details presented in Example 5.

Preparation ofN-Acetyl-2-O-[2-methoxy-4-(2-nitroyinyl)phenyl]-α-D-neuraminic acid (14)

To a stirred solution of compound (13) (25 mg, 0.06 mmol) in a mixtureof ethanol (2.0 mL) and acetic acid (0.02 mL) was added ammonium acetate(24 mg, 0.32 mmol) and nitromethane (0.10 mL, 1.9 mmol) at roomtemperature. The reaction mixture was heated under reflux for 30 min,cooled to room temperature, and evaporated to dryness. The residue waschromatographed (silica gel, 5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride as eluting solvent) to afford pure compound (14) (19mg, 70%): R_(ƒ)=0.64 (5:2:1 ethyl acetate-methanol-0.02% aqueous calciumchloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.07 (s, 3 H, NAc), 1.97-2.04 (m, 1 H, H-3 a), 2.87 (dd,1 H, J_(3e,4) 5.1 Hz, J_(3a,3e) 14.0 Hz, H-3e), 3.54-3.69 (m, 2 H),3.81-4.05 (m, 5 H), 3.93 (s, 3 H, OCH₃), 7.45 (d, 1 H, J 9.6 Hz, ArH),7.48-7.55 (m, 2 H, 2 X ArH), 8.02 (d, 1 H, J 13.3 Hz, H-vinylic), 8.20(d, 1 H, J 13.3 Hz, H-vinylic).

EXAMPLE 7

Preparation of N-acetyl-2-O-(5-bromo-4-chloroindol-3-yl)-α-D-neuraminicacid (28)

The overall reaction scheme is shown in FIG. 12. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

Preparation of 5-Bromo-4-chloro-3-hydroxyindole (26)

To a stirred solution of 5-bromo-4-chloroindoxyl 1,3-diacetate (25) (1.0g, 3.03 mmol) in anhydrous N,N-dimethylformamide (3 mL) was added sodiummethoxide (270 mg, 5.00 mmol). The resulting dark-colored reactionmixture was degassed with nitrogen (g) for 30 min at room temperature.

Preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(5-bromo-4-chloroindol-3-yl)-α-D-neuriminate(27)

The reaction mixture of compound (26) in N,N-dimethylformamide wastreated with stirring with compound (3) (238 mg, 0.468 mmol) at roomtemperature under a nitrogen atmosphere protected from light. After 16h, the reaction mixture was concentrated under vacuum, coevaporated withxylenes (3×25 mL) to remove traces of N,N-dimethylformamide, treatedwith ethyl acetate (40 mL), and filtered. The filtrate was concentratedto a residue that was chromatographed (silica gel, 1:8 acetone-ethylacetate as eluting solvent) to provide compound 27.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

REFERENCES

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1. A chromogenic sialidase substrate compound, having a formula selectedfrom the group consisting of General Structure I; General Structure II,General Structure IIIa; General Structure IIIb; General Structure IVa;General Structure IVb; and of the General Structures, wherein theGeneral Structures are defined as follows:

wherein, R₁═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆,Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₂═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein R₄═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆,Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₅═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂,)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═NO₂, CHO, (CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂, where k is aninteger from 1 to 3, or

wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3;alternatively, for General Structure I, R₁═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₂ or R₄═H, R₆, OR₆,OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO,CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆,OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0to 3; wherein, R₅═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO,CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆,OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0to 3; wherein, R₂ or R₄═NO₂, CHO, (CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂,where k is an integer from 1 to 3, or

wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3;alternatively, for General Structure I, R₁ or R₅═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₂═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₃═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆or CN,where j is an integer from 0 to 3; wherein, R₄═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₁ or R₅═NO₂, CHO,(CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂, where k is an integer from 1 to 3;wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3;

wherein, R₁═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein R₄═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N—OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₅═H or (CH₂)_(k)CH₃, where k is an integer from 0 to 4;wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂;

wherein, R₁═H, OR₃, OC(O)R₄, NO₂, NH₂, N(R₃)₂, Cl, Br, I, F, CHO, CO₂R₃,C(O)N(R₃)₂, C(N—OH)NH₂, OPO₃R₃, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₃, OSO₃R₃,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₃, or CN, where j is an integer from 0 to 3;wherein, R₂═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein R₃═H, C(CH₃)₃,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or (CH₂)_(m)CH₃, where m isan integer from 0 to 3; wherein, R₄═R₃,OR₃, or N(R₃)₂;

wherein, R₁═H, OR₃, OC(O)R₄, NO₂, NH₂, N(R₃)₂, Cl, Br, I, F, CHO, CO₂R₃,C(O)N(R₃)₂, C(N—OH)NH₂, OPO₃R₃, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₃, OSO₃R₃,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₃, or CN, where j is an integer from 0 to 3;wherein, R₂═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₃═H, C(CH₃)₃,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or (CH₂)_(m)CH₃, where m isan integer from 0 to 3; wherein, R₄═R₃, OR₃, or N(R₃)₂;

wherein, R₁═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₆═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₇═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein R₈═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₉═R₈, OR₈, orN(R₈)₂; and

wherein, R₁═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₆═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₇═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N—OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein R₈═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₉═R₈, OR₈, orN(R₈)₂,
 2. The chromogenic sialidase substrate compound according toclaim 1, wherein said compound has the formula of General Structure I ora salt thereof.
 3. The chromogenic sialidase substrate compoundaccording to claim 1, wherein said compound has the formula of GeneralStructure II or a salt thereof.
 4. The chromogenic sialidase substratecompound according to claim 1, wherein said compound has the formula ofGeneral Structure IIIa or a salt thereof.
 5. The chromogenic sialidasesubstrate compound according to claim 1, wherein said compound has theformula of General Structure IIIb or a salt thereof.
 6. The chromogenicsialidase substrate compound according to claim 1, wherein said compoundhas the formula of General Structure IVa or a salt thereof.
 7. Thechromogenic sialidase substrate compound according to claim 1, whereinsaid compound has the formula of General Structure IVb or a saltthereof.
 8. A composition comprising a chromogenic sialidase substratecompound according to claim 1 and a) a solution or b) a dipstick.
 9. Thecomposition according to claim 8, wherein said compound has the formulaof General Structure I or a salt thereof.
 10. The composition accordingto claim 8, wherein said compound has the formula of General StructureII or a salt thereof.
 11. The composition according to claim 8, whereinsaid compound has the formula of General Structure IIIa or a saltthereof.
 12. The composition according to claim 9, wherein said compoundhas the formula of General Structure IIIb or a salt thereof.
 13. Thecomposition according to claim 8, wherein said compound has the formulaof General Structure IVa or a salt thereof.
 14. The compositionaccording to claim 8, wherein said compound has the formula of GeneralStructure IVb or a salt thereof.
 15. The composition according to claim8, wherein the solution is a biological sample.
 16. The compositionaccording to claim 8, wherein the composition farther comprises asialidase.
 17. The composition according to claim 16, wherein thesialidase is bacterial, viral protozoan, or human.
 18. The compositionaccording to claim 16, wherein the sialidase is bacterial.
 19. Thecomposition according to claim 16, wherein the sialidase is viral. 20.The composition according to claim 16, wherein the sialidase isprotozoan.
 21. The composition according to claim 16, wherein thesialidase is human.
 22. The composition according to claim 16, whereinthe sialidase is a vertebrate sialidase.
 23. The composition accordingto claim 15, wherein the biological sample is blood.
 24. The compositionaccording to claim 15, wherein the biological sample is mucosal.
 25. Thecomposition according to claim 15, wherein the biological sample issaliva.
 26. The composition according to claim 8, wherein thecomposition comprises a dip stick.
 27. The composition according toclaim 8, wherein the solution is an aqueous solution.
 28. Thecomposition according to claim 8, wherein the solution is a chemicallyacceptable solvent.
 29. The composition according to claim 8, whereinsaid chromogenic sialidase substrate compound comprises the formula ofGeneral Structure I:

wherein R₁, R₂, R₄, or R₅ are substituents selected from the groupconsisting of H, R₆, Cl, Br, I, F, and NO₂, provided than at least twoof said substituents are substituted with H; R₃ is CH═CHNO₂,

R₆ is H, CH(CH₃)₂, (CH₂)_(m)CH₃ and m is an integer from 0 to 3; orsalts of said chromogenic sialidase substrate compounds.
 30. Thecomposition according to claim 29, wherein R₁ and R₂, are H.
 31. Thecomposition according to claim 29, wherein R₁ and R₄, are H.
 32. Thecomposition according to claim 29, wherein R₁ and R₅, are H.
 33. Thecomposition according to claim 29, wherein R₂ and R₄, are H.
 34. Thecomposition according to claim 29, wherein R₂ and R₅, are H.
 35. Thecomposition according to claim 29, wherein R₄ and R₅, are H.
 36. Thecomposition according to claim 29, wherein R₁, R₂, and R₅ are H.
 37. Thecomposition according to claim 29, wherein R₁, R₄, and R₅ are H.
 38. Thecomposition according to claim 29, wherein R₂, R₄, and R₅ are H.
 39. Thecomposition according to claim 29, wherein R₁, R₂, R₄, and R₅ are H. 40.The composition according to claim 29, wherein R₃ is CH═CHNO₂.
 41. Thecomposition according to claim 29, wherein R₃ is


42. The composition according to claim 29, wherein R₃ is


43. The composition according to claim 29, wherein R₆ is H.
 44. Thecomposition according to claim 29, wherein R₆ is CH(CH₃)₂.
 45. Thecomposition according to claim 29, wherein R₆ is (CH₂)_(m)CH₃ wherein mis an integer from 0 to
 3. 46. The composition according to claim 45,wherein m is
 0. 47. The composition according to claim 45, wherein mis
 1. 48. The composition according to claim 45, wherein m is
 2. 49. Thecomposition according to claim 45, wherein m is 3.